Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells

Abstract

Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease-specific iPSCs have been generated, there has been limited progress in iPSC-based drug screening/discovery for liver diseases, and the low gene-targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency, for which there is currently no drug or gene therapy available, we established a platform to discover new drug candidates and correct disease-causing mutation with a high efficiency. A high-throughput format screening assay, based on our hepatic differentiation protocol, was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients, we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large-scale drug screening, five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition, using the recently developed transcription activator-like effector nuclease technology, we achieved high gene-targeting efficiency in AAT-deficiency patient iPSCs with 25%-33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications.

Conclusions: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs, both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases.

Figure 1. Optimization of the iPSC based hepatic differentiation protocol for high-throughput format drug screening

A A diagram summarizing our modified hepatic differentiation protocol used for the high-throughput format drug screening. Human iPSCs that had been maintained in a feeder free condition were differentiated into hepatic progenitor cells before replating into collagen-1 treated 96-well imaging plates which is specifically designed for a throughput immunofluorescence reader. B Detection of intracellular AAT with a high throughput format immunofluorescence reader. Control iPSC (iH71)- or patient iPSC (iAAT2)- derived mature hepatic cells (10⁵/well) in the 96-well microplates were treated with carbamazepin (CBZ) 5µM for 4 days. AAT staining was performed on the 96-well plates and the AAT fluorescence was obtained using a Safire² microplate reader. RFU: relative fluorescence unit. C Immunofluorescence images of AAT expression obtained from differentiated hepatocyte-like cells of control iPSCs (iH10, left panel) and an AAT-deficiency patient iPSC line (iAAT3, middle panel). Compared to control iPSCs, increased amount of AAT/AAT globules (red) were observed in iAAT cells after hepatic maturation. The expression level was significantly decreased after CBZ treatment (5µM for 4 days, right panel). (×100). D PASD images of these iPSC derived hepatocyte-like cells. Numerous PASD-positive inclusion bodies (pink) were detected within mature hepatocyte-like cells derived from the AAT patient iPSCs (iAAT3, middle panel) but not in the control iPSC (iLC2)-derived hepatocyte-like cells (left panel). The level of PASD+ inclusion body/globules was decreased with CBZ treatment (5µM for 4 days, iAAT3, right panel). (×100).

Figure 2. Patient-specific iPSC based drug screening process and results

A Flowchart depicting the drug screening process and result summary. After the first screening of all the JHDL drugs (3,131 clinical compounds) with the immunofluorescence based screening assay (Fig 1), two hundred sixty-two compounds which significantly decreased accumulation of AAT to less than 50% of the non-treated controls, within the hepatocyte-like cells derived from one AAT-deficient patient iPSC (iAAT2), were selected for further testing. Of the 262 compounds we carefully selected 43 drugs which are relatively safer, without significant unwanted effects to any of the major organs. For the confirmatory screening for the 43 drugs in multiple iPSC lines including iAAT2, iAAT3, iAAT45, and iAAT25, we used independently prepared drugs rather than using the JHDL stock. Of the 43 compounds we discovered 5 final drug hits which consistently showed the AAT reducing effect in the multiple patient derived hepatocyte-like cells. The rest of the drugs did not repeat the results with either one or multiple iPSC lines. B Representative results obtained from multiple independent iPSC lines are shown to demonstrate the AAT reduction effects of the final 5 drug hits in the confirmatory screening (5 µM each for 4 days). *P<0.05, **P<0.01. RFU: relative fluorescence unit. Thi; Thiamine, CBZ; Carbamazepine, Gli; Glipizide, Li; Lithium, VPA; Valproic acid

Figure 3. TALEN-mediated gene correction of AAT mutation in patient-specific iPSCs

A The strategy for precise genome modification using TALEN. Genomic DNA sequence around the Z-mutation (“A” in red) in human AAT gene is shown. A pair of TALEN were designed to specifically recognize the sequences flanking the point mutation with a 16-bp spacer. The targeting vector [homologous recombination (HR) donor] (24) contains the wild-type sequence (“G” in Green) in the homology arm. Selection marker expression cassette PGK-puroΔtk is flanked by piggyBac inverted terminal repeat sequences which allow precise excision upon transient expression of piggyBac transposase. Note that, in order to facilitate this transpose-mediated seamless DNA excision, the endogenous sequence CTGA was changed to TTAA without alteration of the amino acid sequence. Confirmation of gene targeting events by PCR B and Southern blot C. B Genomic DNAs isolated from the picked puromycin-resistant clones after transfection of TALENs and targeting vector were screened by PCR. A 1.2 kb PCR product indicates the targeted events. Using a separate pair of primers, the endogenous allele without targeting event would yield a 1.4 kb band. Lack of such endogenous band would suggest that both alleles were successfully targeted. C Selected clones based on PCR results were confirmed by Southern blot. Parental iPSC (iAAT2) was used as a control. After BamHI digestion of genomic DNA, the endogenous allele without targeting produces a 6.2 kD band. A 4.9-kD band indicates the targeting by the HR donor. D Sequence analysis showing correction of the Z mutation in AAT patient-specific iPSCs after homologous recombination and piggyBac transposase-mediated seamless excision of integrated donor vector. Z mutation and TALEN recognition sequences were shown. While the iPSCs derived from AAT patient showed the G>A mutation (iAAT3), Sanger sequencing of the clone (iAAT3-2) after HR-based gene correction showed wild-type sequence.

Figure 4. Functional restoration of gene-corrected iPSCs

A, B Hepatic differentiation of gene-corrected iPSCs. Gene-corrected iPSC clones were able to differentiate into mature hepatic cells expressing cytokeratin 18 (CK18, A) and albumin (ALB, B). The representative images of a gene-corrected clone (iAAT3-6) derived mature hepatic cells are shown. (×100) C These mature stage hepatic cells derived from the gene corrected iPSCs also exhibit comparable in vitro functional capabilities to healthy control iPSCs as measured by the activities of 4 major cytochrome P450 enzymes (CYP1A2, CYP3A4, CYP2D6 and CYP2C19). D, E PASD staining of mature hepatocyte-like cells derived from a patient iPSC line (iAAT3, D) and the gene-corrected iPSC clone (iAAT3-2, E). (×100) F The levels of AAT detected within the hepatocyte-like cells derived from control-iPSC (iH71), patient-iPSC (iAAT3), and gene-corrected iPSCs (iAAT3-2, red-underlined). The dotted-underlined are the effects of the final drug hits in the screening condition (i.e., 5µM for 4 days) on hepatocyte-like cells derived from the same patient iPSCs.